PARTICIPATION AND STRENGTH OF INTERACTION OF LYSINE 95(BETA) IN THE POLYMERIZATION OF HEMOGLOBIN-S AS DETERMINED BY ITS SITE-DIRECTED SUBSTITUTION BY ISOLEUCINE

The role of Lys-95(beta), which is on the exterior of the hemoglobin ( HbS) tetramer, in the aggregation process has been addressed because t here is a lack of agreement on its importance, The early studies on th e aggregation of HbS in the presence of other mutant hemoglobins are c onsistent with the subsequent electron microscopic studies in demonstr ating the participation of Lys-95(beta) in gelation; the results of th e crystal structure do not agree with these conclusions, Therefore, wi th the objective of clarifying its role we have carried out site direc ted substitution of Lys-95(beta) to an isoleucine residue, The mutatio n was introduced by polymerase chain reaction recombination methodolog y, and the absence of other mutations in the beta-globin gene was esta blished by sequencing the gene in its entirety, The recombinant mutant hemoglobin was expressed in yeast and characterized by peptide mappin g and sequencing, which demonstrated that the only different tryptic p eptide had the Ile substitution at position 95(beta). The recombinant hemoglobin had the correct amino acid composition and molecular weight by mass spectrometric analysis. It was also pure as judged by isoelec tric focusing, It was fully functional because it had an average Hill coefficient of 3.1 and responded normally to the allosteric regulators , chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate, Of par ticular interest was the finding that this hemoglobin mutant aggregate d at a concentration of about 40 g/dl, nearly twice that at which HbS itself aggregated (24 g/dl). Therefore, Lys-95(beta) has a very import ant role in the aggregation process and is a good candidate site for t he design of a therapeutic agent for sickle cell anemia.